Cytotoxicity studies of semi-synthetic derivatives of theveside derived from the aqueous extract of leaves of suicide tree Cerbera odollam

Article abstract of DOI:10.1080/14786419.2014.913242

We report the isolation of two known iridoid glucosides theviridoside (1) and theveside (2) from the aqueous extract of leaves of Cerbera odollam and semi-synthetic derivatisation of theveside prepared in a single step under protection group-free conditions. Derivatives 2a-j were evaluated for cytotoxicity towards five human cancer cell lines of different origins, namely SKBR3 (breast), HeLa (cervical), A375 (skin), HepG2 (liver) and HCT-116 (colon), and IC₅₀ values were determined. Derivatives 2b and 2h exhibited moderate cytotoxicity against HCT-116 and A375 cell lines, respectively.

Full text of DOI:10.1080/14786419.2014.913242

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Cytotoxicity studies of semi-synthetic
derivatives of theveside derived
from the aqueous extract of leaves of
‘suicide tree’ Cerbera odollam
Jaggaiah N. Gorantlaab, Jamsheena Vellekkattab, Lekshmi R.
Nathc, Ruby John Antoc & Ravi S. Lankalapalliab
a Academy of Scientific and Innovative Research (AcSIR), New
Delhi 110 001, India
b Agroprocessing and Natural Products Division, CSIR-National
Institute for Interdisciplinary Science and Technology,
Thiruvananthapuram 695 019, Kerala, India
c Cancer Research Program, Division of Cancer Research,
Rajiv Gandhi Centre for Biotechnology, Thycaud PO,
Thiruvananthapuram 695 014, Kerala, India
Published online: 08 May 2014.
To cite this article: Jaggaiah N. Gorantla, Jamsheena Vellekkatt, Lekshmi R. Nath, Ruby John Anto
& Ravi S. Lankalapalli (2014) Cytotoxicity studies of semi-synthetic derivatives of theveside derived
from the aqueous extract of leaves of ‘suicide tree’ Cerbera odollam, Natural Product Research:
Formerly Natural Product Letters, 28:18, 1507-1512, DOI: 10.1080/14786419.2014.913242
To link to this article: http://dx.doi.org/10.1080/14786419.2014.913242
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Natural Product Research, 2014
Vol. 28, No. 18, 1507–1512, http://dx.doi.org/10.1080/14786419.2014.913242
SHORT COMMUNICATION
Cytotoxicity studies of semi-synthetic derivatives of theveside derived from
the aqueous extract of leaves of ‘suicide tree’ Cerbera odollam
Jaggaiah N. Gorantlaab, Jamsheena Vellekkattab, Lekshmi R. Nathc, Ruby John Antoc and Ravi
S. Lankalapalliab*
aAcademy of Scientific and Innovative Research (AcSIR), New Delhi 110 001, India; bAgroprocessing and
Natural Products Division, CSIR-National Institute for Interdisciplinary Science and Technology,
Thiruvananthapuram 695 019, Kerala, India; Cancer Research Program, Division of Cancer Research,
c
Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram 695 014, Kerala, India
(Received 27 February 2014; final version received 4 April 2014)
We report the isolation of two known iridoid glucosides theviridoside (1) and theveside
(2) from the aqueous extract of leaves of Cerbera odollam and semi-synthetic
derivatisation of theveside prepared in a single step under protection group-free
conditions. Derivatives 2a–j were evaluated for cytotoxicity towards five human
cancer cell lines of different origins, namely SKBR3 (breast), HeLa (cervical), A375
(skin), HepG2 (liver) and HCT-116 (colon), and IC50 values were determined.
Derivatives 2b and 2h exhibited moderate cytotoxicity against HCT-116 and A375 cell
lines, respectively.
Keywords: Cerbera odollam; theviridoside; theveside; semi-synthetic derivatives;
cytotoxicity
1. Introduction
Cerbera odollam, a mangrove plant, belongs to the poisonous Apocynaceae family found along
coastal swamps, riverbanks and creeks in many Asian countries (Gaillard et al. 2004).
C. odollam is also known as Cerbera mangha owing to its striking resemblance with mango tree.
C. odollam is known for prevalence in self-poisoning cases from southern states of India, which
is attributed to about 50% of the plant poisoning cases and 10% of the total poisoning cases in
Kerala, India (Gaillard et al. 2004). The poisonous nature is attributed to cardiac glycosides
known as cardenolides that are present in the latex throughout the plant and seed (Laphookhieo
et al. 2004; Eddleston & Haggalla 2008). Cardiac glycosides are the predominant constituents
from the genus Cerbera, and other chief constituents include lignans, terpenoids, flavonoids and
progesterones (Shen et al. 2007).
In general, pursuit towards phytochemical investigations involves isolation of compounds
from organic solvent extracts, namely n-hexane, ethyl acetate, chloroform and methanol. The
aqueous extracts are usually discarded or less investigated owing to the challenges in purification
and most importantly in characterisation and identification, albeit medicinal plants used in
ayurvedic medicine typically involve aqueous preparations. In this study, we explored the
phytochemical constituents of an aqueous extract from the leaves of C. odollam and isolated two
known iridoid glucosides, theviridoside (1) and theveside (2) (Table 1). The most polar major
constituent isolated was treated with acetic anhydride, and the peracetylated derivative was
found to be sucrose. Theveside (2) with a free carboxylic acid offers an opportunity to build
*Corresponding author. Email: ravishankar@niist.res.in
q 2014 Taylor & Francis

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J.N. Gorantla et al.
Table 1. Structures of theviridoside (1), theveside (2) and semi-synthetic derivatives.
OH OH
O
O
HO
HO
HO
HO
OH
OH
O
O
H
H
OH
OH
O
O
OH
O
OH
O
H3CO
HO
Theviridoside (1)
Theveside (2)
OH
O
HO
HO
OH
O
Method A: BOP, DIPEA, DMF
Method B: HBTU, Et3N, DMF
H
OH
2 + Amine
O
OH
O
R
Entrya
R
Yield (%)b
2a
2b
2c
2d
2e
2f
2g
2h
2i
4-Me-C6H4NH
4-MeO-C6H4NH
4-Cl-C6H4CH2NH
C6H4CH2NH
N,N-Allyl, methyl-N
Butyl-NH
t-Butyl-NH
Cyclohexyl-NH
1-Phenylethyl-1-NH
N,N-Dimethyl-N
50
52
80
55
38
69
68
48
73
48
2j
Abbreviations: BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; HBTU, O-(benzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; DIPEA, N,N-diisopropylethylamine.
a Compounds 2a, 2b and 2j were synthesised by using Method A. Compounds 2c–2i were synthesised by using Method B.
b Isolated yields.
diversity along the skeleton to produce a library of pharmacologically relevant semi-synthetic
derivatives. Accordingly, 10 amide derivatives 2a–j (Table 1) were synthesised in a single step
and screened for cytotoxicity towards five human cancer cell lines. Theveside displayed no
significant cytotoxicity in any of the cancer cell lines tested, while two of its semi-synthetic
derivatives 2b and 2h exhibited moderate cytotoxicity against HCT 116 and A375 cell lines,
respectively.
2. Results and discussion
2.1. Isolation of compounds
The leaf material was dried in a hot air oven between 45 to 508C and then ground to powder
form. To 50 g of the ground plant material, distilled water (1 L) was added and heated between
90 to 1008C for 6 h. After attaining room temperature, the mixture was left to stand overnight and
then filtered. The crude aqueous filtrate was subjected to lyophilisation which afforded 18 g of

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the dried material. A 100.0 mg of lyophilised aqueous extract was purified by reverse-phase
preparative HPLC, for quantification, using the following gradient program: solvent A, H2O;
solvent B, MeOH; linear gradient 0 min 10% B, 35 min 50% B, 40 min 80% B, 45 min 100% B;
detection by charring TLC after spraying with 15% sulphuric acid. Three major products were
isolated and lyophilised to obtain sucrose (41.0 mg), iridoid glucosides theviridoside 1 (4.0 mg)
and theveside 2 (7.4 mg) (Table 1). The NMR results were in close agreement with the literature
(Martin et al. 2007). Iridoids are a group of cyclopenta[c ]pyran monoterpenoids the name of
which was derived from Australian ants, Iridomyrmex, which use these molecules for defence
mechanisms (El-Naggar & Beal 1980; Boros & Stermitz 1991). Iridoids exhibit a wide range of
bioactivities (Tundis et al. 2008), two such iridoid glucosides aucubin and geniposide exert
antitumoral activity by inhibition of topoisomerase I (Galvez et al. 2005).
2.2. Cytotoxicity studies
The in vitro cytotoxicity reports of iridoid glucosides isolated from alcoholic and aqueous
extracts exhibited a good to moderate activity with concentrations in the range of mM against
different human cancer cell lines (Li et al. 2012; Saracoglu & Harput 2012; Rana et al. 2014).
Semi-synthetic derivatives of iridoid glucosides were also reported for in vitro cytotoxicity
Figure 1. Dose-dependent effect of theveside (2) and its semi-synthetic derivatives 2a–j on five cancer cell
lines.

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J.N. Gorantla et al.
studies, which exhibited a better activity than their parent compound (Mouries et al. 2005;
Rakotondramasy et al. 2010). As theveside 2 possesses a carboxylic acid which can be
derivatised to amides, we have synthesised 10 amide derivatives (2a–j, Table 1) in a library-
based approach in a single step with different aromatic and aliphatic amines. Standard peptide
bond coupling agents such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate and O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate
were used for the synthesis under hydroxyl group protection free conditions. In order to
determine the cytotoxicity of theveside 2 and its derivatives 2a–j against various cancer cells,
dose-dependent effect of the compounds on a panel of five cancer cell lines were determined
using the MTT cell viability assay at concentrations ranging from 25 to 200 mM after incubation
for 72 h (Figure 1). A dose-dependent effect on cytotoxicity of the derivatives was clearly
evident from the cell lines with increase of concentration. The IC50 value of compound 2h on
A375 cells was approximately 150 mM, and for compound 2b on HCT-116 was approximately
190 mM (Table S1, Supplementary material). IC50 values of the remaining compounds were
greater than 200 mM. The results clearly indicate that theveside 2 and its derivatives 2a–j did not
affect cell viability of the cervical (HeLa), liver (HepG2) and breast (SKBR3) cancer cells
tested. From a structure–activity standpoint on A375 cells, compound 2h with a cyclohexane
ring is more cytotoxic among the derivatives containing aromatic, aliphatic acyclic and aliphatic
cyclic amides. The MTT results also show that theveside possesses no significant toxicity against
various cancer cell lines, while compound 2h with moderate cytotoxicity indicates that a library
of cyclic aliphatic amides as well as fused and branched bicyclic amides of theveside could
exhibit better cytotoxicity.
3. Experimental
3.1. Plant material
Leaves of C. odollam Gaertn. were collected from Veli lake side, Thiruvananthapuram, Kerala,
India, and were identified by E.S. Santhoshkumar, Jawaharlal Nehru Tropical Botanical Garden
and Research Institute (JNTBGRI), Thiruvananthapuram, Kerala. A voucher specimen (No.
76807) has been deposited in the JNTBGRI Herbarium.
3.2. Cancer cells
Five human cancer cell lines of various origins (SKBR3: breast, HeLa: cervix, A375: skin,
HepG2: liver, HCT-116: colon) were procured from the National Centre for Cell Sciences, Pune,
India. The cells were maintained in DMEM containing 10% FBS and antibiotics at 378C in a
CO2 incubator.
3.3. MTT assay
Cytotoxicity of the synthesised compounds 2a–j along with its parent compound theveside 2
was evaluated by standard MTT assay in five different human cancer cell lines after 72 h of
treatment, as described earlier (Bava et al. 2005). Cells (3000 cells/well) were seeded in 96-well
plates and kept overnight at 378C. The cells were then incubated with different concentrations
(25, 50, 100 and 200 mM) of theveside 2 and its derivatives 2a–j. Curcumin was used as the
positive control. After 72 h of incubation, the percentage of viable cells was determined by MTT
assay. Briefly, after the treatment period, the cells were washed with PBS after removing the
media containing the compounds and then were incubated with fresh medium containing 20%
MTT solution for 2 h. The incubation was continued for another 1 h after addition of extraction
buffer (20% SDS in DMF) to dissolve the newly formed complex. Finally, the optical density

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was measured at 570 nm using a plate reader (Bio-Rad, Hercules, CA, USA) and compared with
that of untreated control. The data were subjected to linear regression analysis for calculation of
IC50 concentrations. Data are average of three independent sets of experiments.
4. Conclusions
The aim of this study was to identify the phytochemical constituents of aqueous extract from the
leaves of poisonous tree C. odollam. We have identified for the first time the presence of sucrose
in large amounts in the leaves along with two known iridoid glucosides theveside 2 and
theviridoside 1. Theveside and its semi-synthetic derivatives 2a–j were tested for in vitro
cytotoxic activity against five cancer cell lines of different origins. Among the compounds
screened, compound 2h displayed the maximum cytotoxic activity in A375 cells (IC50 150 mM)
while most of the compounds did not affect cell viability of the cervical (HeLa), liver (HepG2)
and breast (SKBR3) cancer cell lines. The reason behind this variation in sensitivity of the
cancer cells towards the theveside derivatives is yet to be studied. However, we are trying to
pursue the utility of these derivatives against other disease model cell-based assays.
Supplementary material
Supplementary material relating to this article is available online, including general procedures,
NMR spectral data of theviridoside 1, theveside 2 and synthesis of theveside derivatives 2a–j.
Cytotoxic activity (IC50, mM) of theveside 2 and its derivatives 2a–j. NMR and mass spectra of
theviridoside 1 and theveside 2.
Acknowledgement
This work was financially supported by the Council of Scientific and Industrial Research (CSIR), India,
under the 12th Five-Year Plan (FYP) project NAPAHA.
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